The radiolabeled substrate I-125 N-guanyltyramine was used to identify NAD:arginine ADP-ribosyltransferases in animal tissues. With a purified ADP-ribosyltransferase from turkey erythrocytes using I-125 guanyltyramine (GT) as ADP-ribose acceptor, formation of ADP-ribosyl-I-125-GT was linear with respect to time and enzyme concentration. Transferase activity required NAD; activity with NADP was one-fifth of that with NAD and the reduced pyridine nucleotides were ineffective. The product was indistinguishable on both thin layer and high performance liquid chromatography from that formed by choleragen. ADP-ribosyltransferase activity was demonstrated in crude turkey erythrocyte cytosolic and membrane fractions. When rat liver was fractionated, apparent activity in the standard assay was detected primarily in the microsomes and, like the transferase, was dependent on NAD. The NAD-dependent product of the microsomal reactions was, however, distinguished from the turkey erythrocyte transferase product by thin layer and DEAE-Sephadex chromatography; this product had a retention time identical to that of free I-125 on high performance liquid chromatography. In addition to NAD, the microsomal deiodinase activity was supported by NADH, NADP, and NADPH. Phenyl boronate selectively bound ADP-ribosyl-I-125-GT but not free I-125; however, other metabolites of I-125-GT which were bound by the boronate column were formed by microsomes in a NAD-dependent process. These products could be distinguished from ADP-ribosyl-I-125-GT by high performance liquid chromatography. With liquid chromatographic analysis, I-125-GT can, therefore, be used to quantify ADP-ribosyltransferases in crude mixtures.